When a tissue impairment takes place owing to causation such as a bacterial or viral infection, a physical or chemical trauma, an autoimmune disease, an allergic disease or the like, an inflammatory reaction accompanied with signs such as flare, edema, fever and pain is induced, and accumulation and infiltration of peripheral leukocytes are observed at the local inflammation. The kinds of the leukocytes infiltrating on the site of an inflammation vary depending on the diseases. An acute inflammation such as an ordinary bacterial infection, an immunological complex deposition and a trauma involves accumulation and infiltration mainly of a neutrophile; a tubercular infection, a typhoid infection and a delayed hypersensitivity involve those mainly of a monocyte; and a viral infection involves those mainly of a lymphocyte, while an eosinophile and a basophile infiltrate accompanied with an immediate allergy and a parasite infection [Baggioloni, M. et al., Immunol. Today, 15, 127–133 (1994)]. Recently, there have been found that chemotactic factors of polypeptides having certain degrees of selectivity to leukocytes having chemotactic activities, the polypeptides having characteristic four cysteine residues. Since they are in a family, members of which are homologous in their amino acid sequences and related to each other also in terms of the biological activities, they are referred to as chemokines (having chemoattractant and cytokine activity) [Lindley, I. J. D. et al., Immunol. Today, 14–24 (1993)].
Four cysteine residues of a chemokine are linked with disulfide bonds between the first and third residues and between the second and fourth residues, respectively. Since their characteristics are found on the biological activities in which whether or not one additional amino acid is contained between the first and second cysteine residues, their subfamilies are distinguished by referring to as CXC chemokines and CC chemokines [Baggioloni, M. et al., Adv. Immunol., 55, 97–179 (1994)].
The CXC chemokines which have been found so far are PBSF/SDF-1; IL-8 [Yoshimura, T. et al., Proc. Natl. Acad. Sci. U.S.A., 84, 9233–9237 (1987)]; NAP-2 [Walz, A. et al., Biochem. Biophys. Res. Commun., 159, 969–975 (1989)]; NAP-4; GROα [Richmondo, A. et al., J. Cell. Biochem., 36, 185–198 (1988)]; GROβ [Haskill, S. et al., Proc. Natl. Acad. Sci. U.S.A., 87, 77732–7736 (1990)]; GROγ [Haskill, S. et al., (1990) ibid]; GCP-2 [Proos t, P. et al., J. Immunol., 150, 1000–1010 (1993)]; ENA-78 [Wayz, A. et al., J. Exp. Med., 174, 1355–1362 (1991)]; PF-4 [Deuel, T. F. et al., Proc. Natl. Acad. Sci. U.S.A., 74, 2256–2258 (1977)]; a human CXCR4/fusin/HUMSTSR [Feng, Y. et al., Science, 272, 872–877 (1996)]; and IP-10 [Dewald, B. et al., Immunol. Lett., 32, 81–84 (1992)].
And the CC chemokines are MCP-1 [Yoshimura, T. et al., J. Immunol., 142, 1956–1962 (1989)]; MCP-2 [Chang, H. C. et al., Int. Immunol., 1, 388–397 (1989)]; MCP-3 [Van Damme, J. et al., J. Exp. Med., 176, 59–65 (1992)]; MIP-1α [Obaku, K. et al., J. Biochem., 99, 885–894 (1986)]; MIP-1β [Lipes, M. A. et al., Proc. Natl. Acad. Sci. U.S.A., 85, 9704–9708 (1988)]; RANTES [Schall, T. et al., J. Immunol., 141, 1018–1025 (1988)]; I-309 [Miller, M. D. et al., J. Immunol., 143, 2907–2916 (1989)]; and eotaxin [Jose, P. et al., J. Exp. Med., 179, 881–887 (1994)].
Most of the CXC chemokines have chemotactic activities on a neutrophile but not on a monocyte. Most of the CC chemokines have chemotactic activities on a monocyte but not on a neutrophile. In addition, as to other leukocytes such as an eosinophile, a basophile and a lymphocyte, there have been reported to have the chemotactic activities for some of CXC and CC chemokines. While CC chemokines including RANTES, MIP-1α and MCP-1, and IL-8, which is a CXC chemokine, have been found to possess chemotactic activities on human lymphocytes, none of them are chemotactic factors specific to lymphocytes.
It has been reported that murine PBSF/SDF-1 is a CXC chemokine which is identified as a murine pre-B-cell growth-stimulating factor originally secreted from a murine bone marrow stromal cell line PA6 with its amino acid sequence (FIG. 1) (SEQ ID NO:22), [Nagasawa, T. et al., Proc. Natl. Acad. Sci. USA, 91, 2305–2309 (1994)]. In addition, recently, it has been clarified to have a potent chemotactic activity also on a human T lymphocyte [Bleul, C. et al., J. Exp. Med., 184, 1101–1110].
Various studies have been conducted on receptors for chemokines. There have been reported IL-8RA, which is a receptor specific to IL-8; IL-8RB, which is a receptor for IL-8 and other CXC chemokines; CC CKR1, which is a receptor specific to MIP-1α and RANTES; CC CKR2A, which is a receptor specific to MCP-1; CC CKR2B, which is a receptor specific to MCP-1 and MCP-3; CC CKR3, which is a receptor specific to eotaxin, MCP-3, and RANTES [Combadiere, C. et al., J. Biol., 270, 16491–16494 (1995)]; and CC CKR5, which is a receptor specific to MIP-1α, MIP-1β and RANTES. Recently, CXCR4/fusin/HUMSTSR has been identified as a receptor for SDF-1 which is a human CXC chemokine.
In addition, among the chemokine receptors mentioned above, there has been clarified that CC CKR5, CC CKR2B, CC CKR3 and CXCR4/fusin/HUMSTSR have functions as receptors for HIV-1 by acting cooperatively with CD4, a protein present on a cell membrane, and that an infection with HIV-1 mediated by each receptor is inhibited by the ligands of these receptors.
Two characteristically different HIV-1s are involved in the infection with an HIV-1, which is an AIDS-causing virus, and in the onset of AIDS. A monocyte-tropic HIV-1 with which monocytes, macrophages and T lymphocytes are mainly infected is involved in the viral proliferation in a human body during the period of infection and latent infection, and a T-cell-line-tropic HIV-1 with which T lymphocytes are mainly infected is involved in the reduction of the number of T lymphocytes and the onset of AIDS. In order to infect cells with the two HIV-1s mentioned above, two receptors are required. One is CD4 protein, which is a cell membrane protein, and is a common receptor for the two HIV-1s mentioned above. The other is a protein referred to as a coreceptor which has an activity as a receptor by acting cooperatively with the CD4 protein, and is specific to each of the two HIV-1s.
Recently, there has been clarified that a coreceptor for a main monocyte-tropic HIV-1 is found to be CC CKR5, which is a CC chemokine receptor, and a coreceptor for T-cell-line-tropic HIV-1 is found to be human CXCR4/fusin/HUMSTSR, which is a CXC chemokine receptor. Further, there has been clarified that the infection with a monocyte-tropic HIV-1 is inhibited by MIP-1α, MIP-1β and RANTES, which are CC CKR5 ligands, and the infection with a T-cell-tropic HIV-1 is inhibited by a human PBSF/SDF-1, which is a human CXCR4/fusin/HUMSTSR ligand, suggesting that the chemokine receptors described above could be a target of an HIV-1 infection inhibitor.
On the other hand, which domain in a human CXCR4/fusin/HUMSTSR is essential for the infection with a T-cell-line-tropic HIV-1 has not been identified so far. A CXC chemokine receptor, human CXCR4/fusin/HUMSTSR is a seven transmembrane-spanning-domain receptor, and a three-dimensional structure formed by four extracellular domains is considered to be significant in the binding with a ligand or an HIV-1. For the purpose of identifying a functional domain of a human CXCR4/fusin/HUMSTSR, it is necessary to produce a CXCR4/fusin/HUMSTSR variant so as to maintain the three-dimensional structure as a receptor. The identification of a functional domain of a human CXCR4/fusin/HUMSTSR is extremely useful in the development of an HIV-1 infection inhibitor.
In addition, the elucidation of the mechanism for causation of an HIV-1 species-specificity is significant in the development of an HIV-1 infection model animal, as in the case of clarifying an intracellular factor required for a viral infection. Although a mouse is an excellent experimental animal which can easily be handled and obtained at a low cost of which characteristics are clarified in detail, there has been no report that a mouse is infected with an HIV-1.
Murine cells have several barriers concerning an HIV-1 viral infection. A first barrier is present on the stage where a virus is bound to murine cells. A human CD4 is bound to an HIV-1, but a murine CD4 is not bound to an HIV-1. However, there has been clarified in the existing studies that when a human CD4 is expressed in vitro on the surface of murine cell lines including a T-cell line, the adsorption of an HIV to the cells takes place, but an entry into the cells does not. It was clarified from the above results that the murine cells expressing a human CD4 do not support an entry of the virus, suggesting that there is a receptor, in addition to CD4, which is human-specific and essential for a membrane fusion (which takes place at an entry of the virus) of which molecule is absent in murine cells.
The HIV-1 is recognized to have a difference in an infection ability to CD 4-positive cells depending on the strains. Some strains are classified as monocyte- or macrophage-tropic (M-tropic) strains since they infect a monocyte, and others are classified as T-cell-line-tropic (T-tropic) strains since they infect a T-cell line.
As an HIV-1 infection is progressed, a monocyte-tropic virus observed frequently at an initial stage of the infection is replaced with a T-cell-line-tropic virus. In 1996, there has been reported that CXCR4/fusin, which is a seven transmembrane-spanning-domain G protein-coupled receptor, is essential for the entry of a T-cell-line-tropic HIV-1 into human CD4-positive cells. These results urged the present inventors to study whether or nor the function of the CXCR4 as a viral entry receptor is species-specific.